Functional Group Requirements in the Probable Active Site of the VS Ribozyme

Functional Group Requirements in the ProbableActive Site of the VS Ribozyme

Daniel A. Lafontaine, Timothy J. Wilson, Zheng-Yun Zhao andDavid M. J. Lilley*

Cancer Research UK NucleicAcid Structure Research GroupDepartment of BiochemistryMSI/WTB ComplexUniversity of DundeeDundee DD1 5EH, UK

The VS ribozyme catalyses the site-specific cleavage of a phosphodiesterlinkage by a transesterification reaction that entails the attack of the neigh-bouring 20-oxygen with departure of the 50-oxygen. We have previouslysuggested that the A730 loop is an important component of the activesite of the ribozyme, and that A756 is especially important in the cleavagereaction. Functional group modification experiments reported here indi-cate that the base of A756 is more important than its ribose for catalysis.A number of changes to the base, including complete ablation, leadto cleavage rates that are reduced 1000-fold, while removal of the20-hydroxyl group from the ribose results in tenfold slower cleavage.2-Aminopurine fluorescence experiments indicate that this 20-hydroxylgroup is important for the structure of the A730 loop. Catalytic activity isespecially sensitive to changes involving the exocyclic amine of A756;by contrast, the cleavage activity is only weakly sensitive to modificationat the 7-position of the purine nucleus. These results suggest that theWatson–Crick edge of the adenine base is important in ribozyme func-tion. We sought to test the possibility of a direct role of the nucleobase inthe chemistry of the cleavage reaction. Addition of imidazole base in themedium failed to restore the activity of a ribozyme from which thenucleobase of A756 was removed. However, no restoration was obtainedwith exogenous adenine base either, indicating that the cavity that mightresult from ablation of the base was closed.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: RNA catalysis; functional group modification; acid–basecatalysis; 2-aminopurine fluorescence*Corresponding author

Introduction

RNA catalysis is important in some cellular RNAprocessing reactions,1 – 4 the replication cycles ofsome RNA species,5 – 9 the synthesis of proteins10

and possibly in the splicing of mRNA.11 – 13 Someof the simplest catalytic RNA species are thenucleolytic ribozymes.14 These carry out the site-specific cleavage of a phosphodiester linkage by atransesterification reaction involving attack of theneighbouring 20-oxygen with departure of the50-oxygen. The largest of this class is the VSribozyme, which is found in RNA transcribedfrom the Varkud satellite DNA of Neurosporamitochondria.15

The VS ribozyme is a 154 nt RNA molecule thatacts in cis to cleave itself near to the 50-end (Figure1(a)). It can be divided into a trans-acting ribo-zyme, comprising five helical sections (helicesII–VI), and a substrate stem-loop (helix I) withwhich it interacts by means of tertiary contacts.We have recently deduced the global structure ofthe five-helix ribozyme.16,17 In our model, the over-all geometry is organised by the two three-wayhelical junctions, with an approximately continu-ous axis relating helices IV, III and VI. Helices IIand V extend away from this axis, related by adihedral angle of approximately 758. The ends ofthese helices effectively define the location of thebound substrate. In the cis-acting ribozyme the 30

terminus of the substrate is joined to the 50-end ofhelix II by three nucleotides, while Rastogi et al.18

have identified a long-range interaction betweenthe substrate loop and that of stem V. The twinconstraints suggest that the substrate is located in

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

E-mail address of the corresponding author:[email protected]

Abbreviation used: Rz, ribozyme.

doi:10.1016/S0022-2836(02)00910-5 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 323, 23–34

the cleft between helices II and VI, while making aloop-loop interaction with stem V.

The location of the substrate stem-loop suggeststhat it should make important interactions withhelices II and VI. Analysis of substrate cleavage asa function of sequence changes has highlightedthe loop containing A730 as particularlyimportant,19 such that most single-base substi-tutions lead to large reductions in cleavage rates.The functional importance of this feature is alsoconsistent with results of chemical modificationexperiments.20,21 It is likely that the substratemakes an intimate association with the A730loop,17 and thus this loop is probably the activesite of the ribozyme. Within the A730 loop, onenucleotide stands out. Ribozyme activity isespecially sensitive to changes at A756, and substi-tution by any other nucleotide reduces the rate ofcleavage by three orders of magnitude.19 We havetherefore extended this analysis by carrying out adetailed functional group analysis of A756, fromwhich we conclude that the Watson–Crick edgeof the adenine base is the most critical feature forcatalysis. In the course of these studies we havegenerated a number of fluorescent species that wehave used to probe metal ion-dependent structuralchanges in this region of the ribozyme.

Results

Kinetic analysis of cleavage by a semi-synthetic VS ribozyme

In these studies we have used a trans-acting VSribozyme, where the division between ribozymeand substrate corresponds to our previous ribo-zyme 1 plus substrate 1 combination.16 However,in order to facilitate the introduction of functionalgroup changes at specific positions in the A730loop, we have used a three-piece ribozyme–substrate system shown schematically in Figure2(a). The upper strand of the ribozyme (strand R1)is transcribed, while the lower strand (R2) ischemically synthesised. Strand R2 can be readilyre-synthesised in order to introduce chemically

modified nucleotides at chosen positions. The div-ision of the ribozyme into two unlinked strands isachieved by opening the terminal loop of helix VI;we have shown previously that this results in littleloss of cleavage activity, and that the global foldingof the junction II–III–VI is very similar in isolationor in the context of the complete ribozyme.19

We have made a detailed kinetic analysis of thecleavage activity of the two-part, semi-syntheticribozyme (Figure 2(b)). Radioactively (50-32P)-labelled substrate RNA was incubated with a largeexcess of upper and lower ribozyme strandsat 37 8C in the appropriate reaction buffer. Thecleavage reaction was initiated by addition of mag-nesium ions, and samples were removed at timeintervals and the reaction terminated by chelationof magnesium ions with EDTA. The substrate andthe 5 nt product were separated by polyacrylamidegel electrophoresis, and quantified using digitalphosphorimages. Reaction progress can be wellfitted to a single exponential function (Figure 2(c)),and we have determined an observed rate constantkobs ¼ 0.28 min21 for the bimolecular natural-sequence ribozyme.

Effect of functional group changes at A756 oncleavage activity

We have previously shown that the nucleotidesof the A730 loop are important for catalyticactivity.19 Within the loop, we noted that activitywas especially sensitive to changes at A756, suchthat substitution by any other nucleotide resultedin cleavage rates lowered by three orders of magni-tude. Thus, it is likely that A756 plays an importantrole in the catalysis. We have therefore focused onthis position, and made a range of functionalgroup substitutions, summarised in Figure 3.These include modification to the ribose, functionalgroup changes on the nucleobase and ablation ofthe base. Substrate cleavage has been studied foreach of the modified ribozymes using the three-piece ribozyme–substrate system described above,and the kinetic properties analysed. The resultsfor selected variants are presented in Figure 4, and

Figure 1. The base sequence and secondary structure of the VS ribozyme. (a) The complete sequence of theribozyme,41 with the helices numbered and the position of self-cleavage arrowed. The broken line indicates a proposedtertiary interaction between the loops of helices I and V.18 The nucleotides of the A730 loop are highlighted in boldtype. (b) The local sequence of the A730 loop.

24 The VS Ribozyme Active Site

the kinetic properties of all the species are sum-marised in Table 1.

As we have noted previously,19 removal of the 20-hydroxyl group from the ribose of A756 leads to asmall but significant reduction in the rate of clea-vage. This indicates that this group does not playa critical role in the transesterification reaction. Itis also fortunate, because it extends the range of

modified nucleotides that can be employed inthese studies, as many are available only as20-deoxribonucleotides.

Ablation of the A756 nucleotide base leads to asevere loss of activity. This is true if substituted byan abasic 20-deoxyribose group (930-fold reduced),or simply by a propyl linker (.1000-fold reduced).

Removal of the 6-amino group from the nucleo-base of A756 (i.e. substitution by purine) leads toa large reduction in cleavage activity, and methyl-ation of this group also lowers the catalytic activity.Translocating the exocyclic amine from the 6- to the2-position (2-aminopurine) gives a large reductionin cleavage rate; the same rate was measured forribo-2-aminopurine and 20-deoxyribo-2-amino-purine, indicating that the effects arising from theribose and nucleobase are not additive. The lowactivity of the 2-aminopurine variant could arisebecause the exocyclic amine is required at the6-position, or because it is deleterious at the 2-pos-ition. We therefore replaced the adenine with2,6-diaminopurine, whereupon we obtained asmaller reduction in cleavage activity. Thissuggests that the larger effect arises from the lossof the amine from the 6-position, consistent withthe low activity of the A756Purine variant.

We have also investigated the importance of theN7 position in the purine nucleus. Replacement ofthe sp 2-hybridised nitrogen by a C–H (7-deaza-adenosine) leads to almost complete retention ofnormal cleavage activity. Thus, despite the impor-tance of A756 in the activity of the VS ribozyme,its N7 is of no consequence in the cleavagereaction.

Analysis of KMapp and k2 for A756

variant ribozymes

We have further analysed the cleavage reactionfor selected variants as a function of ribozymeconcentration (e.g. Figure 4(c)). This is analysedon the assumption of the formation of a non-covalent complex between the ribozyme (Rz) andsubstrate (S), i.e.:

Rz þ SOk1

k21

Rz·SOk2

k22

Rz·P1·P2!k3

Rz·P2 þ P1

where P1 and P2 are the products. k2 is the rate ofthe central conversion of the bound substrate intoproduct. In addition to the chemical step, it mayinclude conformational changes, such as arearrangement of substrate secondary structure.22

The short 50 reaction product should rapidly dif-fuse away, and thus k22 can be ignored, since k3 qk22: The data are fitted to the equation:

kobs ¼ ðk2½Rz�Þ=ð½Rz� þ KappM Þ ð1Þ

From this we have determined the values of KMapp

and k2. We have previously measured values of1.0 mM and 2.0 min21, respectively, for the natural-sequence, one-piece ribozyme. The values for theA756 variants (Table 2) show that, in general, the

Figure 2. Kinetic analysis of the VS ribozyme variants.(a) Schematic of the substrate plus two-piece ribozymeconstruct. This corresponds to a substrate 1 þ ribozyme1 type trans-acting system, where the substrate and ribo-zyme are separated by dividing the RNA between A639and G640. In general this gives relatively rapid, mono-phasic cleavage kinetics. The ribozyme is itself dividedinto two RNA species in this construct, strand R1(black) and strand R2 (grey). This is achieved by remov-ing the hairpin loop at the end of helix VI. (b) Substratecleavage by the natural sequence two-piece ribozyme.Radioactively (50-32P)-labelled substrate (,1 nM) wasincubated with an excess of ribozyme (1 mM) at 37 8C inthe presence of 10 mM magnesium ions. The substrateand product were separated by polyacrylamide gelelectrophoresis, and quantified by phosphorimaging.(c) The progress of the cleavage reaction is plotted asa function of time, and fitted to a single exponential toobtain the rate.

The VS Ribozyme Active Site 25

effects of the functional group substitutions arelargely due to impairment of the cleavage rate k2,and that substrate binding is relatively unaffected.

Effect of functional group changes at C755 andG757 on cleavage activity

We have also examined the effect of functionalgroup changes on the nucleotides flanking A756in the A730 loop. Cleavage activity was measuredusing the three-piece ribozyme–substrate system,and the kinetic properties analysed (Table 1).

Removal of the 20-hydroxyl groups from eitherposition lead to almost undetectable changes insubstrate cleavage rate (,1.5-fold reduced in bothcases). Replacement of C755 by 2-aminopurineresulted in a 65-fold reduction in cleavage rate,while the corresponding replacement at G757 leadto a 4.3-fold decrease in rate. Thus ribozyme func-tion is less sensitive to changes in nucleobase andribose for C755 and G757 compared to A756.

Fluorescent properties of 2-aminopurinesubstituents in the A730 loop

2-Aminopurine is fluorescent, but its fluor-escence is strongly quenched by the stacking ofadjacent bases, so changes in the intensity of fluor-escence are indicative of changes in the localenvironment of the fluorophore.23 – 26 We have

therefore exploited the availability of VS ribozymevariants containing this base at various positionswithin the A730 loop to make a spectroscopicstudy of conformational changes induced by mag-nesium ion binding. Magnesium ion bindingwithin this region has been suggested by the sensi-tivity of ribozyme activity to phosphorothioatesubstitution in the loop, and the restoration ofactivity in the presence of thiophilic manganeseions.21 In our experiments, we have used the two-piece ribozyme (corresponding to strands R1 andR2 in Figure 2(a)), with either ribo-2-aminopurineor 20-deoxyribo-2-aminopurine, and studied thefluorescence intensity as a function of magnesiumion concentration. Representative spectra areshown in Figure 5(a). For each of the substitutedribozymes we observe significant changes in fluor-escence intensity and anisotropy on addition ofmagnesium ions, from which we can estimateapparent magnesium-binding parameters assum-ing a simple two-state ion-induced transitionmodel (see Materials and Methods).

Fluorescence intensity as a function of mag-nesium ion concentration is very similar for riboseand 20-deoxyribose forms of G7572AP (Figure5(b)); fitting to the two-state model resulted in aslightly higher affinity for magnesium ions for theribose form ([Mg2þ]1/2 is 370 mM compared to580 mM). The intensity increases approximately2.5-fold and the anisotropy increases from 0.15 to

Figure 3. Summary of functional group modification of adenine 756. Schematic to show the different base and ribosemodifications used in the analysis of the role of A756 in the cleavage reaction. For the base modifications, the sugarring is indicated by R; this is either ribose or 20-deoxyribose as indicated in Table 1.


Figure 4. Effect of functionalgroup modification at A756 on sub-strate cleavage. (a) Substrate clea-vage by A756 variant ribozymes.Radioactively (50-32P)-labelled sub-strate (,1 nM) was incubated withan excess of two-piece ribozyme(1 mM) for 30 minutes in the pre-sence of 10 mM magnesium ions.Substrate and product were sepa-rated by gel electrophoresis asbefore, and visualised by phosphor-imaging. Track 1, substrate alone.Track 2, substrate incubated withnatural sequence ribozyme. Tracks3–8, substrate incubated withribozyme in which A756 has beensubstituted by 20-deoxyribose-7-deazaadenine, 20-deoxyribose-N 6-methyladenine, 20-deoxyribodiamino-purine, 2-aminopurine, 20-deoxy-ribopurine and a propyl spacer,respectively. (b) Progress curves forcleavage reactions using selectedA756-variant ribozymes. Plots ofcleaved fraction as a function oftime for 20-deoxyribose-7-deazaade-nine variant (diamonds), 20-deoxy-riboadenine (circles), 20-deoxyri-bose-N 6-methyladenine (invertedtriangles), 20-deoxyribodiamino-purine (triangles), ribo-2-amino-purine (squares). (c) The cleavagereaction as a function of ribozymeconcentration for the A756 purinevariant. Rate constants for substratecleavage were measured for a rangeof ribozyme concentrations. Thesedata are plotted, and fitted toequation (1). The resulting valuesof k2 and KM

app are presented inTable 2, along with correspondingvalues for other A756 variants.


0.23 with addition of magnesium ions, whichtogether indicate that the base loses flexibility andis held in a position where adjacent bases do notstrongly quench the fluorescence. The Hill coef-ficient is n ¼ 1.7, indicating that cooperative bind-ing of more than one magnesium ion influencesthe structure of the A730 loop.

In contrast with the effects at position 757, a sig-nificant difference in the effect of magnesium on2-aminopurine fluorescence is observed for theribose and 20-deoxyribose forms at position 756,consistent with the effects of removal of the20-hydroxyl group on ribozyme activity. The fluor-escence intensity of ribo-2-aminopurine decreaseson addition of magnesium ions, with [Mg2þ]1/2 ¼620 mM and n ¼ 1.4. By contrast, the intensity of20-deoxyribo-2-aminopurine increases on additionof magnesium ions, with [Mg2þ]1/2 ¼ 1.1 mM andn ¼ 0.83. These data suggest that the 20-hydroxyl

group contributes to the affinity for magnesiumions.

The intensity of fluorescence was significantlyweaker for 2-aminopurine at position 755 (datanot shown), probably due to quenching by thetwo adjacent purines.26 – 28 Consequently, it is notpossible to obtain reliable estimates of apparentmagnesium ion-binding parameters. However, theaffinity for magnesium ions appears to be reducedin this variant, consistent with the impairedactivity of the C755 2-aminopurine ribozyme.

The low sensitivity of ribozyme activity to2-aminopurine substitution at position 757suggests that we can exploit the G757 2-amino-purine variant as a reporter of structural changeselsewhere in the A730 loop, and we have usedthis to assess the conformational consequences ofribose modification at positions 755 and 756(Figure 5(c)). 20-Deoxyribose substitution at C755greatly perturbs the local environment of 2-amino-purine at position 757, resulting in a markedchange in the response of the fluorescence intensityto magnesium ion concentration. Similarly, a20-deoxyribose substitution at position 756 signifi-cantly affects the fluorescence of 2-aminopurine atposition 757. The addition of a 20-O-methyl groupto A756 lowered the apparent affinity for mag-nesium ions but did not alter the end-points of thetitration. As was found for the 20-deoxyribose-2-aminopurine at position 756, the Hill coefficientis less than one, suggesting that the 20-hydroxylat position 756 is necessary for the binding of amagnesium ion.

Significance of the adjacent base-pair C731–G754 on cleavage activity

We have also examined the effect of changes tothe junction-distal flanking base-pair on theactivity of the ribozyme. In our previous studiesof the sequence requirements in stem VI we foundthat the majority of sequence alterations outsidethe A730 loop lead to rather small changes inactivity, except for certain changes in the C731–G754 base-pair. We considered the possibility thatthese bases might be unpaired, thereby extendingthe A730 loop in that direction. We thereforecarried out a systematic variation of these twonucleotides. For these experiments, we used ourconventional ribozyme 1 plus substrate 1 system,where the terminal loop of helix VI is intact.16 Thekinetic results are summarised in Table 3.

Single base changes lead to a loss of substratecleavage activity of between 26 and 83-fold. Thesechanges necessarily disallow the formation of aWatson–Crick base-pair. Much smaller changes inactivity were obtained when the double substi-tutions C731A:G754U and C731U:G754A weremade; these replace the C–G base-pair that closesthe A730 loop with A–U and U–A base-pairs.Taken together, these results do not support thedisruption of the C731–G754 base-pair in theactive ribozyme.

Table 1. Effect of functional group changes at variouspositions in the A730 loop on cleavage activity

Variant kobs (min21) kred

Natural sequence 0.28 (1)

A. A756 position20-Deoxyribosea 0.029 9.720-Deoxy abasic 0.0003 930Propyl linker ,0.0001 .280020-Deoxyribopurine 0.0001 280020-Deoxyribo N 6-methyl 0.0034 8220-Deoxyribo 2-aminopurine 0.0002 1400Ribo 2-aminopurine 0.0002 140020-Deoxyribo diaminopurine 0.0024 12020-Deoxyribo 7-deaza 0.075 3.7

B. C755 position20-Deoxyribose 0.19 1.520-Deoxyribo 2-aminopurine 0.019 15Ribo 2-aminopurine 0.0043 65

C. G757 position20-Deoxyribose 0.17 1.620-Deoxyribo 2-aminopurine 0.0085 33Ribo 2-aminopurine 0.065 4.3

kobs was measured for the cleavage reaction in trans, using thetwo-piece ribozyme system under standard conditions. kred isthe factor by which a given variant is impaired relative to thenatural sequence, i.e. kobs (natural)/kobs (variant).

a Data from Lafontaine et al.19

Table 2. Kinetic parameters for A756 ribozyme variants

Variantk2

(min21)KM

app

(mM)k2/KM

app

(min21 mM21)

Natural sequence 2.0 1.0 2.020-Deoxyadenine 0.30 5.1 0.0620-Deoxy purine 0.0012 7.2 0.000220-Deoxy 2-aminopurine 0.0013 5.5 0.0002

Ribozyme titrations were performed for selected variants,from which k2 and KM

app were measured. The second-order rateconstant k2/KM

app was calculated from these values. Asymptoticstandard deviations on the fits indicate that errors in k2 andKM

app are generally less than 10% of values.


Inactivity of A756 abasic variant in thepresence of imidazole and adenine

One possible reason for the strong sensitivity ofVS ribozyme activity to modifications of the base

of adenine 756 is that the nucleobase might play adirect role in the chemistry of the cleavage reac-tion, perhaps by general acid–base catalysis. Wehave therefore tested the possibility that additionof imidazole base to the medium restores the

Figure 5. Fluorescent propertiesof A730 loop 2-aminopurine vari-ants. (a) Fluorescence spectra(lex ¼ 312 nm) showing the emis-sion from 2-aminopurine in theG757 ribo2-aminopurine variant asa function of magnesium ionconcentration in the range 0.025–50 mM. The fluorescence intensityincreases with each addition ofmagnesium ions. (b) Integratedfluorescence intensity (arbitraryunits) of 2-aminopurine plotted as afunction of magnesium ion concen-tration for variant ribozymes.The lines show the fits obtainedusing a simple two-state model(see Materials and Methods), yield-ing the parameter estimates listedbelow. The ribozyme variantsshown are ribo-2-aminopurineincorporated at positions 756 (filledcircles; n ¼ 1.4, [Mg2þ]1/2 ¼620 mM), and 757 (filled triangles;n ¼ 1.7, [Mg2þ]1/2 ¼ 370 mM), and20-deoxyribo-2-aminopurine incorpo-rated at positions 756 (open circles;n ¼ 0.83, [Mg2þ]1/2 ¼ 1.1 mM), and757 (open triangles; n ¼ 1.7,[Mg2þ]1/2 ¼ 580 mM). (c) Ribo-2-aminopurine was incorporated atposition 757, and its integratedfluorescence intensity (arbitraryunits) measured as a function ofmagnesium ion concentration inthe presence of a second substi-tution elsewhere in the A730 loop.The second substitutions were20deoxyribocytosine 755 (invertedtriangles), 20deoxyriboadenine 756(open triangles) and 20-O-methyl-riboadenine 756 (circles). The 20-O-methyl-riboadenine 756 data werefitted using the two-state model,yielding parameter estimates ofn ¼ 0.83 and [Mg2þ]1/2 ¼ 730 mM.The broken line shows the fit of thetwo-state model to the data forribo-2-aminopurine at position 757shown in (b).


activity of A756 variants. Convincing evidencefor acid–base catalysis by cytosine 75 in the HDVribozyme has been obtained by the demonstrationof restoration of activity of a C75U variant byaddition of exogenous imidazole base.29

We have studied the activity of some stronglyimpaired A756 variants of the VS ribozyme in thepresence of buffers containing high concentrationsof imidazole, using the trans-acting two-pieceribozyme. We observed no detectable restorationof activity with A756U, A756C or A756G variants(data not shown). This might be a result ofhindered access into the required region of theactive site in the presence of the variant nucleotide.We have therefore repeated the experiment usingthe A756Abasic ribozyme (Figure 6). However, wefailed to detect any restoration of activity underthe conditions used in these experiments; indeed,activity was fourfold lower in the presence ofimidazole (as it was for the natural sequence ribo-zyme). The experiments were also repeated usingthe propyl spacer, also with negative results (datanot shown). In further experiments, we also failedto observe restoration of cleavage activity of the

A756Abasic ribozyme in the presence of exogenouscytosine, isocytosine, 2-aminopyridine, 2-amino-pyrimidine, 2,6-diaminopurine or 4-amino-pyrimidine (data not shown). The failure of theexogenous imidazole to restore activity mightmean that A756 is not active in acid–base catalysis.However, it could simply be a result of collapse ofthe cavity formed in the abasic ribozyme, andthus a failure of the base to gain access. In anattempt to distinguish these possibilities we incu-bated substrate and A756Abasic ribozyme in thepresence of adenine base; once again little or norestoration of activity was detected. We concludethat removal of the base of A756 (or replacementof the entire nucleotide by three carbon atoms)probably leads to a structural rearrangement ofthe active site that prevents access by exogenousbases under these conditions.

Discussion

The A730 loop is probably the most importantpart of the VS ribozyme from a functional point ofview, and should be regarded as a major com-ponent of the active site. Within this loop, themost significant role is likely to be played byadenine 756. Replacement of A756 by any othernucleotide leads to a major loss of activity, withoutaltering the folding of the ribozyme at least at aglobal level. By contrast, cleavage rates are moretolerant of substitution of other nucleotides in theloop. Moreover, the effects of substitutions at A756on cleavage activity are largely manifested at thelevel of the cleavage rate (k2) rather than bindingaffinity (KM

app). The A730 loop also exhibits bindingof divalent metal ions. Ribozyme activity isreduced by phosphorothioate substitution at phos-phates 757 and 758, but activity is restored in thepresence of manganese ions.21 Uranyl ion-inducedcleavage has also been observed at C755 inan RNA duplex containing the A730 loop (C.Hammann, D.A.L. & D.M.J.L., unpublished data).

Table 3. Effect of base substitutions at the C731:G754base-pair on cleavage activity

Sequence kobs (min21) kred

Natural sequence 1.0 (1)C731A 0.015 67C731G 0.039 26C731U 0.039 26G754A 0.025 40G754C 0.012 83G754U 0.022 45C731A:G754U 0.12 8.3C731U:G754A 0.30 3.3

kobs was measured for the cleavage reaction in trans, using aone-piece ribozyme system (ribozyme 1 plus substrate 116)under standard conditions. kred is the factor by which a givenvariant is impaired relative to the natural sequence, i.e. kobs

(natural)/kobs (variant).

Figure 6. Cleavage reactions per-formed using A756 abasic ribozymein the presence of exogenous bases.Substrate cleavage by the naturalsequence two-piece ribozyme withand without addition of exogenousimidazole and adenine. In eachcase, radioactively (50-32P)-labelledsubstrate (,1 nM) was incubatedwith an excess of ribozyme (1 mM)at 37 8C in the presence of 10 mMmagnesium ions. The reaction wasterminated after five minutes usingthe natural sequence ribozyme, or60 minutes using the A756 abasic

ribozyme. Substrate and product were separated by polyacrylamide gel electrophoresis, and visualised by phosphor-imaging. Tracks 1–3, natural sequence; tracks 4–6, A756 abasic ribozyme. Tracks 1 and 4, reaction in 40 mM Tris–HCl (pH 8.0); tracks 2 and 5, reaction in 200 mM imidazole (pH 7.5); tracks 3 and 6, reaction in 3 mM adenine,40 mM Tris–HCl (pH 8.0).


Our new results reinforce our view that A756has a very important role in the catalysis of thecleavage, since some relatively subtle changesto functional groups lead to large reductions insubstrate cleavage rates. Catalytic activity in theVS ribozyme is especially sensitive to changesinvolving the exocyclic amine of A756. Its removal,translocation to the 2-position and even its methyl-ation result in large reductions in cleavage rate.By contrast, the cleavage activity is almost totallyinsensitive to modification at the 7-position of thepurine nucleus. Taken together, these resultssuggest that the Watson–Crick edge of the adeninebase is important for the cleavage reaction.

We can envision two reasons why VS ribozymeactivity is so sensitive to changes at A756. Theadenine base could have a structural role, perhapsforming a base-pair with the substrate, since pre-vious studies have shown that some changes atA756 do not affect ribozyme folding.19 However,no essential uridine is present in the internal loopof the substrate,22 and therefore any interactionmust be non-Watson–Crick. Furthermore, wehave compared the kinetic parameters of three20-deoxyribose variants at position 756; all threehave closely similar values of KM, yet the ribozymecontaining an adenine base has a rate of cleavage(k2) that is 300 times faster than those with either2-aminopurine or purine. Therefore, the presenceor absence of the 6-amino group does not signifi-cantly contribute to substrate binding. It remainspossible that changes at A756 result in local confor-mational changes in the active site that impair thecleavage process.

The alternative is that the base might participatedirectly in the cleavage reaction. For example, N1might act as a general base to remove a protonfrom the attacking 20-hydroxyl in order to improveits nucleophilicity. This would require the elevationof its pKa to a value that is closer to neutrality, butthis might be possible within the environment ofthe folded active site, and elevated pKa valueshave been measured for adenine bases within thelead-zyme by means of NMR.30 However, thereare two pieces of evidence that appear to argueagainst a role in general acid–base catalysis.

First, the rate of substrate cleavage by the VSribozyme is essentially independent of pH.31 Thisindicates that there is no proton transfer in therate-determining step. However, the chemicalcleavage step may not be rate-determining; forexample the proposed rearrangement of substratestructure22 might be slow enough to mask thechemical step. A similar situation has been foundin the hairpin ribozyme.32 Additionally, there is nosimple relationship between the pKa of modifiedbases introduced at position 756 and ribozymeactivity.

Perhaps the best characterised example ofnucleobase participation in RNA-mediated cataly-sis has been found in the HDV ribozyme. The crys-tal structure33 shows that an essential cytosine ispoised by the substrate cleft. Substitution of this

cytosine to guanine or uracil leads to a large lossin activity, but this can be partially restored byaddition of exogenous base such as imidazole.29

By contrast, we have been unable to recovermeasurable cleavage activity by addition ofexogenous imidazole base to an abasic substitutionat position 756, providing the second potentialargument against general acid–base catalysis byA756. Yet while base rescue experiments of thiskind are very convincing when they give positiveresults, a negative result does not necessarilyexclude such a role for the endogenous nucleobase.In the case of a substitution of one base for another,physical access to the required site may not bepossible. When an abasic site is generated, theresulting cavity may collapse, altering the localstructure of the active site. This is supported byour failure to restore activity by addition ofexogenous adenine base. Nucleobase rescueexperiments have been carried out exhaustively inthe core of the hammerhead ribozyme, wherelarge reductions in cleavage rate were observedwith abasic substitutions.34 – 36 However, activitywas only restored in a subset of these, most ofwhich were clustered where the domain 2 scaffoldabuts the end of stem II; this region is likely tohave a greater natural rigidity that might with-stand the formation of an abasic cavity. By contrast,ablation of the nucleobase from A756 in the centreof the A730 loop may conceivably result in amajor perturbation of the structure, from whichrescue by exogenous base or nucleobase is notpossible.

The base of A756 appears to be more importantthan its ribose for catalysis. Thus, while a numberof changes to the base lead to cleavage rates thatare reduced 1000-fold, removal of the 20-hydroxylgroup from the ribose results in tenfold slowercleavage. While the latter change is significant,and is larger than the effects resulting from thecorresponding changes at C755 or G757, it is muchlower than the effects of base modifications atA756. We believe that this functional group isimportant for the structure of the A730 loop butdoes not play a direct role in catalysis, since substi-tutions have a relatively small effect on k2. Theweaker affinity for magnesium ions and the lackof cooperativity in the binding isotherms suggestthat the 20-hydroxyl group at position 756 is neces-sary for the binding of at least one divalent metalion. A 20-deoxyribose substitution at position 756has recently been shown to reduce activity in anucleotide analogue interference analysis of theVS ribozyme.37

In summary, it is clear that the base of A756plays an important role in the catalytic activity ofthe VS ribozyme, while its ribose probably contri-butes to the folding of the A730 loop into thecorrect structure. Analysis of functional groupchanges indicates that the Watson–Crick edge ofA756 is the most critical part of the base, and mayparticipate in general acid–base catalysis. How-ever, this remains conjecture at the present time;


further definition of the role will probably requirestructural analysis of the A730 loop, and suchstudies are in hand.

Materials and Methods

RNA synthesis

Oligonucleotides containing both DNA and RNA sec-tions were synthesised on a 200 nmol scale using phos-phoramidite chemistry38 implemented on an AppliedBiosystems 394 DNA/RNA synthesiser. Ribonucleosideb-cyanoethyl phosphoramidites were obtained fromPrOligo as rA(4-(t-butylphenoxy)-acetyl) (tac), rG(tac),rC(tac) and U with 20-t-BDMS protection. DCI (PrOligo)was used as an activator. The capping A reagent wasreplaced by (tac2O) (PrOligo). Extended coupling wasused for non-standard nucleotides (purine, N 6-methyl-adenine, 7-deaza-adenine, 2-aminopurine, 2,6-diamino-purine, propyl and deoxyribose) (Glen Research), withaverage step-wise coupling yields of 99%. Oligoribo-nucleotides were deprotected in 25% ethanol/ammoniasolution at 55 8C for four hours, and evaporated to dry-ness. They were dissolved in 0.5 ml 1 M tetrabutylammo-nium fluoride (Aldrich) in tetrahydrofuran to removet-BDMS groups, and agitated at 20 8C in the dark for 16hours prior to desalting by G25 Sephadex (Pharmacia)and ethanol-precipitation. Fully deprotected oligonucleo-tides were purified by electrophoresis in 20% (w/v)polyacrylamide gels containing 7 M urea. Bands wereexcised, and oligonucleotides electroeluted into 8 Mammonium acetate and recovered by ethanol-precipi-tation.

Transcription of RNA

RNA was transcribed from double-stranded DNAtemplates using T7 RNA polymerase.39 Templates fortranscription of ribozymes were made by recursive PCRfrom synthetic DNA oligonucleotides. RNA was purifiedby electrophoresis in 8% or 20% polyacrylamide gelscontaining 7 M urea. RNA was recovered from crushedgel slices by elution in water at 4 8C overnight. ElutedRNA was filtered, recovered by ethanol-precipitationand dissolved in water.

Analysis of ribozyme cleavage

Ribozyme cleavage reactions were performed usingtrace concentrations of radioactively (50-32P)-labelled sub-strate and an excess of two-piece ribozyme. Substrate(,1 nM) and 1 mM of each ribozyme strand were mixedin the presence of 0.1 mM EDTA and heated at 90 8C forone minute before addition of 40 mM Tris–HCl (pH8.0), 1 mM EDTA, 2 mM spermidine, 25 mM KCl in atotal volume of 20 ml. For the experiments performedusing exogenous bases (Figure 6), the Tris buffer waseither replaced with 200 mM imidazole (pH 7.5) orsupplemented with 3 mM adenine base. The cleavagereaction was initiated by addition of 11 mM MgCl2 and2 ml aliquots were removed at different times, and thereaction terminated by addition of 8 ml of 95% forma-mide, 20 mM EDTA, 0.05% (w/v) xylene cyanol FF,0.05% (w/v) bromophenol blue. Substrate and productwere separated by electrophoresis in a 20% poly-acrylamide gel containing 7 M urea, and quantified byexposure to a storage phosphor screen and imaging

(Fuji). Data were fitted to single exponential functionsby regression analysis using the initial part of the pro-gress curve (Kalaidagraph, Abelbeck Software).

Fluorescence spectroscopy

Fluorescence spectroscopy was performed on anSLM-Aminco 8100 fluorimeter, and spectra were cor-rected for lamp fluctuations and instrumental variationsas described.40 Excitation and emission polarisers werecrossed at 54.78. All data were collected at 4 8C in10 mM Tris–HCl (pH 8.0), 25 mM KCl. Spectra werecorrected for background and the effect of dilution overthe course of a titration, and intensities were determinedby integrating the data collected over the range 330–450 nm. Data for 2-aminopurine fluorescence as a func-tion of magnesium ion concentration were fitted to thesimple two-state model:

I ¼ I0 þ DI £KA½Mg2þ�n

1 þ KA½Mg2þ�nð2Þ

where I is the fluorescence intensity, I0 is the intensity inthe absence of magnesium ions, DI the change inintensity, KA is the apparent association constant for thebinding of magnesium ions and n is a Hill coefficient.As the parameters KA and n covary, we present the[Mg2þ]1/2 ¼ (1/KA)1/n, which gives a robust estimate ofthe affinity for magnesium ions. Values of fluorescenceanisotropy (r ) were determined from:

r ¼ ðFVV 2 GFVHÞ=ðFVV þ 2GFVHÞ ð3Þ

where G is correction factor given by the ratio FHV/FHH

and the subscripts V and H refer to fluorescence withvertical and horizontal polarisers, respectively, in theorder excitation, emission.

Acknowledgements

We thank David Norman for discussion, CancerResearch UK and BBSRC for financial support andEMBO and the Canadian Institutes of HealthResearch for fellowships awarded to D.A.L.

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Edited by J. Karn

(Received 25 June 2002; received in revised form 19 August 2002; accepted 21 August 2002)


Functional Group Requirements in the Probable Active Site of the VS Ribozyme

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Transcript of Functional Group Requirements in the Probable Active Site of the VS Ribozyme